Journal of Cerebral Blood Flow & Metabolism (2012) 32, 33–40& 2012 ISCBFM All rights reserved 0271-678X/12 $32.00
www.jcbfm.com

Cerebral oxygen metabolism of rats using
injectable 15O-oxygen with a steady-state method
Masato Kobayashi1,2, Tetsuya Mori1, Yasushi Kiyono1, Vijay Narayan Tiwari1,
Rikiya Maruyama1, Keiichi Kawai1,2 and Hidehiko Okazawa1,3
1

Biomedical Imaging Research Center, University of Fukui, Fukui, Japan; 2School of Health Sciences,
College of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Ishikawa, Japan;
3
Research and Education Program for Life Science, University of Fukui, Fukui, JapanTo develop a less-stressful and simple method for measurement of the cerebral metabolic rateof oxygen (CMRO2) in small animals, the steady-state method was applied to injectable 15O2-PET(15O2-positron emission tomography) using hemoglobin-containing vesicles (15O2-HbV). Ten normal
rats and 10 with middle cerebral arterial occlusion (MCAO) were studied using a small animal PET
15
scanner. A series of 15O-PET scans with C15O-labeled HbV, H15
O2-HbV were performed with
2 O, and10 to 15 minutes intervals to measure cerebral blood volume (CBV), cerebral blood flow (CBF), and
CMRO2. Positron emission tomography scans were started with a tracer injection using a
multiprogramming syringe pump, which provides a slowly increasing injection volume to achieve
15
O2-HbV scans. The radioactivity concentration of 15O rapidly
steady-state radioactivity for H15
2 O and15
achieved equilibrium in the blood and whole brain at about 2 minutes after H15
O2-HbV
2 O andadministration, which was stable during the scans. The whole brain mean values of CBF, CBV, and
CMRO2 were 54.3±2.0 mL per 100 g per minute, 4.9±0.4 mL/100 g, and 2.8±0.2 lmoL per g per
minute (6.2±0.4 mL per 100 g per minute) in the normal rats, respectively. In the MCAO model rats,
all hemodynamic parameters of the infarction area on the occlusion side significantly decreased.
The steady-state method with 15O-labeled HbV is simple and useful to analyze hemodynamicchanges in studies with model animals.
Journal of Cerebral Blood Flow & Metabolism (2012) 32, 33–40; doi:10.1038/jcbfm.2011.125; published online 24 August 2011
Keywords: 15O-gas PET; cerebral oxygen metabolism; hemoglobin-containing vesicles; injectablesteady-state method

Introduction
15

O-gas positron emission tomography (PET) has
been used to clarify the mechanisms of cerebrovascular diseases (CVDs) using PET (Temma et al, 2006,2008). An intravenous injectable 15O2 method was
proposed using a special device and the blood wascollected from another rat because radioactivityof 15O-gas from the airways affects measurement of
cerebral radioactivity counts (Magata et al, 2003;
Temma et al, 2006). Our previous study reported thefeasibility of another injectable 15O2 method using
artificial hemoglobin-containing vesicles (HbV),
Correspondence: Professor H Okazawa, Biomedical Imaging
Research Center, University of Fukui, 23-3 Matsuoka-Shimoaizuki,
Eiheiji-cho, Fukui 910-1193, Japan.
E-mail: okazawa@u-fukui.ac.jp
This study was partly funded by a Grant-in-Aid for Scientific
Research from Japan Society for the Promotion of Science(19790861, 22791180) and the Fukui Brain Project, Research and
Education Program for Life Science, University of Fukui.
Received 29 April 2011; revised 27 July 2011; accepted 28 July
2011; published online 24 August 2011

15

O2;

which does not require killing of other rats forblood collection and the special device (Tiwari et al,2010). Since these injectable 15O2 methods appliedbolus injection of tracers to measure cerebral
hemodynamic parameters, multipoint arterial blood
samplings to obtain arterial input functions wererequired, which may be stressful for small animals
and may change physical conditions. Therefore, wedeveloped a new steady-state method for cerebral
blood flow (CBF) measurement using H15
2 O anda multiprogramming syringe pump that provides
a slowly increasing injection volume to reduce rats’stress (Kobayashi et al, 2011). The steady-statemethod is ideal for small animal studies because
the injection volumes and blood samplings arelimited.
The purpose of this study was to apply the steadystate method to 15O2-labeled HbV (15O2-HbV) PET
scans to measure the cerebral metabolic rate ofoxygen (CMRO2). C15O-labeled HbV (C15O-HbV)
PET was also performed to obtain the cerebral blood
volume (CBV), which is usually used for correctionof the oxygen extraction fraction (OEF).

Injectable 15O2 steady-state method
M Kobayashi et al
34

Materials and methods
Labeling Procedure of Hemoglobin-Containing Vesicles
The synthesis and purification processes of HbV (Oxygenix
Co. Ltd., Tokyo, Japan) and rheological properties of HbV
were described previously in detail (Phillips et al, 1997;
Ogata, 2000; Takeoka et al, 2002; Tiwari et al, 2010).
L-cysteine (2.8 mM) was added to HbV vials, which were
kept under anoxic conditions. The HbV was labeled with
C15O and 15O2 by the method reported previously (Tiwari
et al, 2010). The HbV solution of 2 mL for C15O labeling and
8 mL for 15O2 labeling was prepared in a 50-mL volume
vial. Target gas containing C15O or 15O2 was delivered into
the HbV solution through a bubbling needle. The bubblingtime of the radioactive gas was 2 minutes, and the vial
was vortexed during 15O2 bubbling. Labeled HbV was thensampled using a syringe and radioactivity was measured
using a dose meter (Capintec Inc., Ramsey, NJ, USA).

Animal Positron Emission Tomography Studies
Animal studies were approved by the Animal Care
Committee at the University of Fukui and conducted inaccordance with the international standards for animal
welfare and institutional guidelines. Adequate measures
were taken to minimize pain and discomfort. Male
Sprague-Dawley rats from Japan SLC Inc. (Hamamatsu,
Japan) were housed for 1 week under a 12-hour light/
12-hour dark cycle with free access to food and water. The
rats were fasted with no food overnight with watersupplied ad libitum before the PET experiments.
Twenty rats (298.5±8.8 g) were anesthetized with an
intraperitoneal injection of chloral hydrate (0.4 mg/g bodyweight, intraperitoneally). A PE-50 catheter was inserted
into the femoral artery for blood sampling and the femoral
vein for 15O-radiotracers administration. In 10 rats, the left
middle cerebral artery (MCA) was occluded intraluminallyusing a nylon 4-0 surgical monofilament (Ethilon; Ethicon,
Inc., Somerville, NJ, USA) for 1 hour before the PET scans(Longa et al, 1989; Kuge et al, 1995). The PET studies were
performed with a list-mode scan protocol using a small
animal PET scanner (SHR-41000; Hamamatsu Photonics,
Hamamatsu, Japan) (Yamada et al, 2008). The scanneracquires 213 slices covering an axial length of 160 mm,
with a three-dimension mode, achieving a resolution of
B2.0 mm full width at half maximum in the transaxial
direction and 2.8 mm full width at half maximum in theaxial direction. The rats were placed in a supine positionon the scanner bed, and the limbs were fixed using surgicaltape. The orientation of the cranial position was determined using a laser beam on the scanner. During the PET
scan, body temperature was maintained at about 371C
using a controller (TC-1, Brainscience idea, Osaka, Japan).
Before emission scans, a transmission scan was performed
for attenuation correction using a 68Ge/68Ga externalsource. A 3-minute PET scan was started with an
intravenous bolus administration of about 20 MBq C15OHbV in a 0.4-mL injection volume. Approximately 10 mL ofarterial blood was sampled every 1 minute, and radioactivity in the blood samples was immediately measured
Journal of Cerebral Blood Flow & Metabolism (2012) 32, 33–40

with a well scintillation counter (ARC380; Aloka, Tokyo,
Japan). After the decay of C15O, a 5-minute scan was started
with intravenous administration of B185 MBq H15
2 O. The
3-mL tracer was injected using a 10-mL syringe set up on amultiprogramming syringe pump (FP-2000; Melquest,
Toyama, Japan). Details of this steady-state method were
described in our previous report (Kobayashi et al, 2011).
About 50 mL arterial blood was sampled every 1 minuteduring the H15
2 O scan, and radioactivity in the blood
samples was immediately measured with the well scintillation counter. After the decay of H15
2 O, a 5-minute PET
data acquisition was started with intravenous administration of B185 MBq 15O2-HbV in a 3-mL volume using thesame injection method as for the H15
2 O scan. Briefly, theinjection speed was controlled rapidly for the first 5
seconds to fill the dead volume of the venous line with asmall amount of overshoot, the rate was then changed
moderately for 40 seconds to increase blood radioactivity,
followed by a slow but gradually increasing administration
rate to compensate for the decay in blood 15O radioactivity.
To compensate for the decay in blood 15O radioactivity
in the later phase, the injection rate was continuously
changed under the assumption of an inverse decayfunction as expressed by the following equation:
v ¼ 117  expð1:0102  l  tÞ

ð1Þhere v (mL/min) is the injection velocity for the syringe
pump, l (/min) is the decay constant of 15O, and t (minutes)
is the time after the injection of 15O-radiotracers. The same
constants were used in the equation for both 15O2-HbV andH15
2 O administration. About 200 mL arterial blood was
sampled every 1 minute during the 15O2-HbV scan. In all,
50 mL of the arterial blood was put into a microtube tomeasure whole blood radioactivity. In all, 50 mL acetonitrile, a deproteinization agent, was added into the remaining arterial blood and the sample was centrifuged at 3000 g
for 2 minutes. About 100 mL of supernatant fluid including
acetonitrile was obtained, and radioactivity was immediately measured with a well scintillation counter.
Arterial blood gases and other physiological parameters
were measured using a blood gas analyzer (ABL555;
Radiometer, Copenhagen, Denmark) and a hemooximeter(OSM3; Radiometer) after the transmission scan, andbefore and after the 15O2-HbV PET scan.

Magnetic Resonance Imaging Scan and
2,3,5-Triphenyl-Tetrazolium Chloride Staining
After PET scans, the normal rats and 3 of the 10 MCA
occlusion (MCAO) model rats were scanned by magnetic
resonance imaging (MRI) using a 3.0-T MRI scanner (Signa
Horizon, GE Medical Systems, Milwaukee, WI, USA).
In the MRI scans, the rats were placed and fixed between
a pair of surface coils. T2-weighted images of each rat’s
brain were acquired using a fast spin echo methodwith repetition time/echo time of 5050/85 milliseconds,
256  256 matrix, slice thickness 1.0 mm, spacing 0 mm,
field of view 10 cm, phase-field of view 8 cm and excitation
number of 3. The rat brains were removed immediately
after the MRI scans, sliced and stained with 2,3,5-

Injectable 15O2 steady-state method
M Kobayashi et al35

triphenyl-tetrazolium chloride (TTC). The remaining sevenrats with MCAO were killed immediately after the PET
scans, and the brains were sliced and stained with TTC.

Data Analysis
The PET images were reconstructed using the Fourier
rebinning-filtered back projection method with an attenuation correction, calibrated by the crosscalibration factor.
The CBV image was calculated using PET data averaged forthe last 2 minutes of the 3-minute C15O-HbV scan because
the time–activity curve of the brain achieved a plateau
at about 1 minute after the tracer administration. The
radioactivity concentration of the arterial blood sampled
every 1 minute after the C15O-HbV injection was averaged
and used for the CBV calculation. The CBF was calculated
from H15
2 O images using the steady-state method asreported previously (Kobayashi et al, 2011). The average
image of the last 3 minutes data of the 5-minute H15
2 O-PETscan was used for the CBF calculation.
Since the radioactivity concentrations of the brain
and arterial blood reached a plateau about 2 minutes after
15
O2-HbV administration (see Results), the average of thelast 3 minutes data of the 5-minute 15O2-HbV scan wereused for OEF and CMRO2 calculations. The OEF image was
15
calculated from the averaged images of H15
O2-HbV
2 O andscans based on the established steady-state methodwith the following equations (Frackowiak et al, 1980;
Lammertsma et al, 1982);
OEF

0

CbO2 CaH2O
CaH2O
CbH2O  CpO2  CpH2O
¼
CaO2
CaH2O
CpO2  CpH2O

CaH2O =CpH2O ¼ 1  0:245Hta

ð2Þ

ð3Þ

where OEF’ is uncorrected OEF, CbO2, and CbH2O (Bq/mL)
are the average brain radioactivity concentration of the 15O2
and H15
2 O images. CaO2, CpO2, CaH2O, and CpH2O (Bq/mL)
are the mean arterial and plasma concentrations in the 15O2
and H15
2 O scans, respectively. Hta is arterial hematocrit. The
CBV correction method was applied to the calculationof OEF from OEF’ as follows (Lammertsma et al, 1983;
Lammertsma and Jones, 1983).
OEF ¼

CBF=100þl
OEF 0  CBF=CBVþl
CBF=100þl
1  CBF=CBVþlð4ÞIn the equation (4), l is the decay constant of 15O.
The CMRO2 was calculated by the following equation
using the CBF, OEF images, and arterial O2 content (O2c),which was obtained from the blood gas and hemoglobin
data of each rat.
CMRO2 ¼ OEFCBFO2 c

ð5Þ

The parametric PET images of normal rats were coregistered with each MRI image using Dr View software (AJS,
Tokyo, Japan). In the analysis of normal rats, MRI coronalslices were used to draw ROIs (regions of interest) at threeslice levels of the whole brain, frontal cortex, visual cortex,
striatum, thalamus, and sensorimotor cortex referring to
the rat brain atlas (Kobayashi et al, 2011). The same ROIs

were applied to all parametric PET images to obtain
cerebral hemodynamic parameters in each normal rat.
In the MCAO model rats, multiple circle ROIs (2.4 mm indiameter) were placed on the infarction area and thecorresponding contralateral regions with reference to the
TTC staining brain slices, as well as the MRI images in thethree MCAO model rats. The hemodynamic parameters of
the affected cortex in the MCAO model rats were comparedwith the corresponding contralateral region and those of
the sensorimotor cortex in the normal rats using one-way
analysis of variance with a post hoc Sheffe’s F-test. Bloodgas data in the normal and MCAO model rats werealso compared using repeated-measures analysis ofvariance and a post hoc paired t-test. A P value of < 0.05
was considered statistically significant.

Results
Labeling Efficiency and Physiological Parameters
15
Labeling efficiency values of H15
2 O, C O-HbV, and15
O2-HbV were 123.4±3.4, 65.7±12.2, and95.7±4.1 MBq/mL, respectively.
Table 1 shows arterial blood gas data after thetransmission scan, and before and after the 15O2-HbV
PET in the normal and MCAO model rats. Only PCO2
values of the MCAO model rats decreased significantly for all conditions compared with normal rats
(P < 0.05), although no rat group showed any difference among conditions (repeated-measures analysis
of variance). The injection volume of the 15O2-HbV
and H15
2 O was 1.65 mL during the 5-minute PET
scan. The sampled blood volumes were about 0.65
15
O2-HbV scans,and 1.40 mL for the H15
2 O andrespectively.

Normal Rat Study

The 15O2-HbV radioactivity concentration rapidly
achieved equilibrium in the whole blood, plasma,
and the brain at about 2 minutes after radiotracer
administration using the same injection program as
the H15
2 O-PET (Figure 1), and the curves were similar
to those of H15
2 O. Representative PET images of anormal rat are given in Figure 2. Table 2 shows
hemodynamic parameters of the whole brain mean
and the regional cerebral values in the normal rats.
The parameters were not significantly different
among the brain regions.
Middle Cerebral Arterial Occlusion Model Rat Study

The MCAO model rats also showed rapid radioand 15O2-HbV in
activity equilibrium of H15
2 O
the brain and blood within 2 minutes using thesteady-state method. Figure 3 shows MRI images,
TTC staining, and 15O-PET images in a MCAO model
rat. The TTC staining brain slice revealed an
infarction area in the left cerebral hemisphere after
MCAO. Cerebral hemodynamic parameters of the
Journal of Cerebral Blood Flow & Metabolism (2012) 32, 33–40

Injectable 15O2 steady-state method
M Kobayashi et al
36

Table 1 Arterial blood gas data in normal and MCAO model rats
Before 15O2-HbV

After transmission

pH
PCO2 (mm Hg)
PO2 (mm Hg)
Hct (%)
O2 Sat (%)
Hb (g/dL)
O2c (mLO2/g)

After 15O2-HbV

NormalMCAONormalMCAONormalMCAO

7.31±0.04
47.8±2.2
88.2±4.0
48.0±2.3
96.7±1.2
15.1±0.5
0.20±0.01

7.31±0.03
44.0±2.6*87.0±6.0
49.2±2.0
95.8±2.3
15.5±0.8
0.21±0.01

7.32±0.06
48.6±1.588.1±5.847.3±2.3
95.4±2.1
15.1±0.4
0.20±0.01

7.32±0.06
39.9±3.8*88.0±5.6
49.5±1.8
94.7±2.3
15.5±0.6
0.21±0.01

7.32±0.05
49.0±1.1
90.5±6.547.3±2.0
97.0±1.3
15.1±0.6
0.20±0.01

7.32±0.06
42.2±3.7*87.1±7.948.3±1.794.9±3.915.6±0.6
0.21±0.01

ANOVA, analysis of variance; Hb, hemoglobin; Hct, hematocrit; MCAO, middle cerebral arterial occlusion; O2 Sat, arterial oxygen saturation; O2c, O2 content;
PCO2, arterial carbon dioxide tension; PO2, arterial oxygen tension.
*P < 0.05 compared between normal rats and MCAO rats for each condition by ANOVA. The three different conditions did not affect blood gas data in
any group (repeated-measures ANOVA).

Radioactivity (kBq/ml)

500
400
300
200
100
0
0

1

2
3
Time (min)4

5

Figure 1 Time–activity curves of the whole blood, plasma, andthe whole brain using 15O2-HbV with the steady-state method.
The radioactivity reached equilibrium in the whole blood (K),plasma, (’) and the whole brain (J) rapidly at B2 minutes
after administration. HbV, hemoglobin-containing vesicles.

normal rats and the MCAO model rats are given in
Table 3. The hemodynamic parameters in thecontralateral side were not significantly different
from those in the sensorimotor cortex of the normal
rats. All hemodynamic parameters in the ipsilateralhemisphere decreased significantly compared withthe contralateral side (P < 0.01).

Discussion
In this study, the steady-state method was applied
to 15O2-HbV PET to measure the cerebral oxygen
metabolism of rats under normal and MCAO conditions, providing rapid radioactivity equilibrium inboth the blood and the brain. The physical conditionof rats was stable during PET experiments because of
the small volumes of injection as well as arterial
blood samplings. Injectable 15O2-HbV is ideal formeasuring the cerebral oxygen metabolism withsmall animals because 15O2-gas inhalation affects
Journal of Cerebral Blood Flow & Metabolism (2012) 32, 33–40

cerebral PET counts due to its high nasal radioactivity. Although cerebral oxygen metabolism of
normal rats was estimated using the 15O2-HbV witha single bolus injection method (Tiwari et al, 2010), a
small animal may be stressed from frequent multipoint arterial blood samplings in each 15O experiment. In our previous study, the steady-state method
was applied to measure CBF using H15
2 O andprovided stable values. The method was considered
suitable for repeated CBF measurements because it
requires only a few arterial blood samplings during
the steady state of the PET scan (Kobayashi et al,2011).
The same injection program was applied to 15O2HbV administration in the present study. Although
the labeling efficiency of the 15O-radiotracers
15
O2-HbVwas slightly different between H15
2 O and(123.4 and 95.7 MBq/mL), the difference did notaffect the time to achieve equilibrium of radioactivity
in the blood and the brain because the same level ofradioactivity of 61.7 MBq/mL for both radiotracers(185 MBq in 3 mL volume) was injected at the scanstart. The injection volume of the 15O-radiotracer did
not affect the physical condition of the rats during
the PET experiments (Table 1) because of the small
injection volume (1.0 and 0.25 mL net injection
15
O2-HbV, respectively, after
volumes for H15
2 O andsubtraction of the sampled blood volumes) andslowly increasing injection velocity (Morton et al,1997). In addition, HbV injection did not change therats’ physical condition. Although the PCO2 values in
the MCAO model rats were significantly decreased
compared with those in the normal rats, probably
due to hyperventilation in a few rats during PET
experiments, the CBF was not affected significantly
because other blood gas data were not changed
compared with those of normal rats. This effect of
MCAO operation on the arterial blood gas data
was similar to a previous report (Temma et al,2006). The PCO2 values seem slightly higher and thePO2 values seem slightly lower in normal rats
compared with those in humans probably due to thetendency of hypoventilation under anesthesia duringInjectable 15O2 steady-state method
M Kobayashi et al
37

L

R

15
Figure 2 Coronal magnetic resonance imaging (MRI) (A), C15O-HbV (B), H15
O2-HbV (D) positron emission tomography
2 O (C), and
15(PET) images of a normal rat. Average PET images of 2 minutes for C15O-HbV, and 3 minutes for H15
O2-HbV after reaching
2 O andequilibrium were used to calculate the hemodynamic parameters. Each PET image was coregistered to the individual MRI, and sliced
at the same level. The upper values for the color scale were 50, 300, and 300 (kBq/mL) for (B), (C), and (D), respectively. HbV,
hemoglobin-containing vesicles.

Table 2 Hemodynamic parameters of normal rats
Whole
brain

FrontalcortexVisualcortexStriatum

ThalamusCBF (mL per minute 100 g)

54.3±2.054.1±1.853.8±2.1

55.1±2.2

56.2±1.8

OEF

0.56±0.04

0.53±0.05

0.56±0.05

0.55±0.04

0.56±0.05

CMRO2 (mmoL per minute per g)(mL per minute per 100 g)

2.78±0.19(6.2±0.4)

2.60±0.21(5.8±0.5)

2.73±0.20(6.1±0.4)2.72±0.19(6.1±0.4)

2.82±0.21(6.3±0.5)

CBV (mL/100 g)

4.9±0.4

5.1±0.5

5.1±0.4

4.8±0.4

5.0±0.5

CBF, cerebral blood flow; CBV, cerebral blood volume; CMRO2, cerebral metabolic rate for oxygen; OEF, oxygen extraction fraction.
See values for the sensorimotor cortex in Table 3.

L

R

Figure 3 Transaxial magnetic resonance imaging (MRI) (A), 2,3,5-triphenyl-tetrazolium chloride (TTC) staining (B), H15
2 O (C),15
O2-HbV (D), and C15O-HbV (E), positron emission tomography (PET) images on a left middle cerebral arterial occlusion (MCAO)
model rat. All images are shown at the same slice level, including the sensorimotor cortex, etc. The regions of interest (ROIs) wereplaced on the infarction in the ipsilateral side and on the normal cortex in the contralateral side (A). Dashed lines are contours of thebrain. The upper values for the gray scale were 300, 300, and 50 (kBq/mL) for (C), (D), and (E), respectively. HbV, hemoglobincontaining vesicles.
Journal of Cerebral Blood Flow & Metabolism (2012) 32, 33–40

Injectable 15O2 steady-state method
M Kobayashi et al
38

Table 3 Hemodynamic parameters of the rats under normal and MCAO conditions
Normal (n = 20)

MCAO (n = 10)

Sensorimotor cortex

Contralateral

Ipsilateral

CBF (mL per minute per 100 g)

55.8±1.856.2±1.923.8±2.6*

OEF

0.56±0.04

0.54±0.03

0.24±0.04*

CMRO2 (mmol per minute per g)(mL per minute per 100 g)

2.86±0.21(6.4±0.5)

2.88±0.19(6.4±0.4)

0.55±0.13*(1.2±0.3*)

CBV (mL/100 g)

5.0±0.4

5.3±1.0

2.8±0.5*

ANOVA, analysis of variance; CBF, cerebral blood flow; CBV, cerebral blood volume; CMRO2, cerebral metabolic rate for oxygen; MCAO, middle cerebral artery
occlusion; OEF, oxygen extraction fraction.
*P < 0.0001 compared among the three groups (20 hemispheres of normal rats and 10 of contra- and ipsi-lateral hemispheres of MCAO rats) for eachhemodynamic parameter using ANOVA and post hoc Sheffe’s F-test.

experiments. Similar results were observed in aprevious study with normal rats (Tiwari et al, 2010).
In the present study, OEF images were correctedusing CBV images obtained from the C15O-HbV,
providing accurate cerebral oxygen metabolism.
This blood volume correction has never been appliedin small animal studies. The CBV correction isnecessary because the CBV is usually changed in
the affected area of a CVD model animal. The CBVimages were obtained using data from 1 to 3 minutes
after C15O-HbV administration because cerebral
counts reached a constant level at about 1 minuteafter administration. The hemodynamic parametersin the contralateral side of the MCAO model rats
were not different from those in the normal rats.
However, the parameters of the ipsilateral infarction
area showed significant decreases (Table 3), because
the MCAO model in the present study caused
complete infarction in the brain territory of theoccluded MCA. Since complete occlusion andinfarction would prevent the ability of the tracers
to reach the lesion, the MCAO model in this study
may affect the time to achieve equilibrium andoxygen membrane permeability in the ischemic
tissue, which may influence on quantitative values
obtained by the steady-state method. This significant
reduction of parameters in the region of cerebral
infarction was similar to a previous report (Temmaet al, 2006). A single-compartment analysis used in
the steady-state method may overestimate cerebral
water clearance and tissue counts (Ohta et al, 1992,
1996); however, this method was established inhuman PET studies and many studies with CVD
patients were reported (Yamauchi et al, 1996, 1998,
1999; Okazawa et al, 2001; Ouchi et al, 2001).
Although slightly different values were observed
between the steady-state method and the bolus
administration method in a previous study withCVD, the values were not significantly different(Okazawa et al, 2001).
The 15O2-HbV injection yields metabolic 15O-water,which may affect arterial blood and cerebral PET
counts. However, the radioactivity of the metabolic
water was considered to reach equilibrium in the
Journal of Cerebral Blood Flow & Metabolism (2012) 32, 33–40

early phase after 15O2-HbV administration because
the plasma radioactivity in the 15O2-HbV scanreached a plateau at 2 minutes after administrationin the present study using the steady-state method(Figure 1). The cerebral radioactivity of H15
2 O-PET
also achieved equilibrium at 2 minutes after administration as reported previously. The average images
15
O2-HbV were obtained using PET dataof H152 O andfrom 2 to 5 minutes after administration because
cerebral PET counts and arterial blood radioactivity
were constant during that time. Stable hemodynamic
parameters were obtained with the same quality andnoiseless PET images, as well as stable radioactivityof the arterial blood and plasma (Table 2). Although
the steady-state method may induce systemic underestimation of CBF because of tissue heterogeneity
between gray and white matter as compared withthe bolus injection method in the human study(Kanno et al, 1984); however, the structure of thebrain cortex in rats is not so complicated (Zilles andWree, 1995), and the effect of brain tissue heterogeneity is not considered so significant as comparedwith the human brain because we placed ROIs on thecortices (Figure 3A) and basal ganglia. The cerebral
hemodynamic parameters of the normal rats using
the present steady-state method were similar to theresults reported previously using the bolus method(Magata et al, 2003; Temma et al, 2006; Tiwari et al,2010), in which slightly higher values of OEF and
CMRO2 in normal rats were observed comparedwith those of normal human subjects. Differencesin species and effects of anesthesia during theexperiments are considered to provide these differences. Higher CBV values may also be caused byanesthesia, and many previous reports showedvarious rage of CBV from 2.5 to 4.7 mL/100 g (Sandor
et al, 1986; Todd et al, 1993; Perles-Barbacaru andLahrech, 2007). A smaller size of HbV than normal
RBC may also have affected the CBV value because
smaller vessels might be delineated by C15O-HbV.
The blood pool correction using CBV was applied inthis steady-state method, and thus, the influence ofslightly greater vascular radioactivity on OEF values
would be negligible.

Injectable 15O2 steady-state method
M Kobayashi et al

A small amount of 15O2-gas may be detached from15
O2-HbV in the lungs and exhaled, which may affectradioactivity in the head and airways of rats during
PET scans. However, it has already been reported thatthe 15O2-gas released from the nose and mouth
showed little influence on quantitative values of
cerebral oxygen metabolism in previous studies
using injectable 15O2 (Magata et al, 2003; Temmaet al, 2006). Although the radioactivity of 15O2 released
from the nose and mouth of the rats in 15O2-HbV
studies has never been measured, the influence of
detached 15O2 on quantitative hemodynamic values
would be similarly small, as in previous studies. The
penumbra regions were not examined in this study
because a reperfusion model after MCAO was notapplied and most of the ischemic regions showed
infarction. A reperfusion model after MCAO would
be appropriate to evaluate the pathophysiology of the
penumbra area. In the rats with MRI images, thebrains were excluded about 15 to 20 minutes after the
PET scans. There was a slight time difference fromthe procedure without MRI in the timing of the TTC
staining; however, the infarction area in the TTC
staining was not significantly different from that in
the PET and MRI images (Figure 3).
In conclusion, 15O2-HbV with the steady-statemethod is stable and useful, and may be ideal tomeasure the cerebral oxygen metabolism of small
animals with less stress. The CBV correction with
C15O-HbV provides precise cerebral oxygen metabolism, especially in CVD model animals. Basic
research with CVD models will be accelerated byinjectable 15O-HbV tracers with the steady-statemethod.

Acknowledgements
The author thanks Satonao Nakakoji, Akira Ito,
Hiroshi Oikawa, and other staff of the Biological
Imaging Research Center, University of Fukui for
their technical support.

Disclosure/conflict of interest
The authors declare no conflict of interest.

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